No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

The large geophytic monocotyledon, Hypoxis colchicifolia Baker, has been identified for the importance of its corm extracts in the development of a potential non-toxic prodrug for the treatment of inflammation, certain malignancies and HIV-infection. The underground corms of this plant are also commonly used for therapeutic applications in traditional medicine in Kwazulu-Natal where it primarily occurs. A review of published literature revealed, however, that H. colchicifolia plants are currently harvested in an unsustainable manner from traditional collecting sites due largely to population growth, increased land use for urban development and agriculture, and the popularisation of Hypoxis plants for herbal remedies. A further search of historical records established that H. colchicifolia plants were dominant in grassland vegetation prior to 1950, but had rapidly declined since then. Quantitative data subsequently gathered in this study from comparative surveys of both H. colchicifolia and H. hemerocallidea populations from sites with near-pristine, disturbed, burnt and mown grassland vegetation showed for the first time that exposure to human activity and the grassland management practices of mowing and burning incurred not only a 75% reduction in plant density of both these Hypoxis species, but also the total destruction of mature plants of H. colchicifolia in frequently mown and burnt areas. Flowering data recorded in these surveys, and confirmed by monitoring field performance of cultivated H. colchicifolia plants, showed that a contributing factor to the plant's inability to withstand these pressures was that juvenile forms only reached flowering maturity after three to four years growth, thus adversely affecting seedling recruitment. It was concluded therefore that, since Hypoxis species responded differently to mowing and burning, geophytic plants should be considered individually and not as "forbs" during the planning of grassland management programmes for natural conservation areas. The need to cultivate H. colchicifolia to ensure its survival was also established using the new field data gathered in this study. Methods to propagate this species have, however, not been established. Data gathered on all the plants comprising a single population confirmed that mature plants survive to an estimated 20 years and longer in natural areas. Greatest hypoxoside yields were also obtained from corms with a fresh mass of 350g to 400g. Since these corms were estimated to be 10-years-old and older, propagation and cultivation methods that could sustain plant production and survival for long periods, and therefore increased hypoxoside yields, would have to be developed. Several micropropagation systems suitable for the mass production of H. colchicifolia and from which phenotypically normal plantlets were recovered, were therefore established via organogenesis, embryo culture and somatic embryogenesis. The latter cultures have not been reported previously for Hypoxis. In the former culture the toxic effects of phenolic leachates and browning were controlled, and improved plantlet regeneration achieved, by adding polyvinyl pyrrolidone to the medium and introducing distinct sequential aseptic steps into the micropropagation procedure developed. Defined protocols for the different phases of in vitro somatic embryogenesis are not readily available for monocotyledons, however, neither are the factors controlling embryogenesis and organ regeneration known. In this study the process of somatic embryogenesis from excised zygotic embryos of H. colchicifolia was shown to be complex and the resultant cultures very heterogeneous. Although the stage of development of the zygotic embryo explants was important at the time of inoculation, data showed that the induction and regulation of the processes of embryo culture and somatic embryogenesis were ultimately determined by the exogenously applied plant growth regulators. By comparing the different pathways leading to plantlet regeneration, and the morphological stages of development of the structures produced both on solid and in liquid media, not only photographically, but also quantitatively and schematically, the repeated formation of pseudoembryonic structures and neomorphs confirmed that they form an integral part in the in vitro somatic embryogenic pathway of H. colchicifolia. Evidence suggested not only that two types of somatic embryos are produced in the embryogenic cultures of H. colchicifolia, but that the pseudoembryonic structures produced resemble the pseudobulbils produced in polyembryonic cultures of Citrus. The success of the somatic embryogenic cultures was confirmed by the estimation that 28 112 somatic embryos and embryo clusters of H. colchicifolia could be obtained from 16 ml of somatic embryogenic liquid culture. Furthermore phenotypically normal plantlets regenerated from all of the micropropagation procedures developed were successfully transplanted from the laboratory, acclimatized under greenhouse conditions and their horticultural and field performances evaluated. / Thesis (Ph.D.)-University of KwaZulu- Natal, Pietermaritzburg, 2004.

The wide utilization and popularity of medicinal plants in African Traditional Medicine (ATM) has been recognized and attributed to the effectiveness, affordability and accessibility of these medicinal plants. However, the extensive exploitation of medicinal plants has exacerbated the strain on the wild populations. In vitro propagation/micropropagation is an effective method which allows for mass production or multiplication of pathogen-free plants that are morphologically and genetically identical to the parent plant. In addition, the technique is contributing to the understanding of metabolic pathways and regulating the production of plant secondary products. Eucomis autumnalis (Mill.) Chitt. subspecies autumnalis (Hyacinthaceae) is a valuable medicinal species in ATM and commonly traded in the urban street markets of South Africa. Currently, the conservation status of this species has not been evaluated. However, as with most bulbous plants, the wild population is continuously under threat due to over-harvesting and habitat loss via various anthropogenic factors. Thus, in vitro propagation is a viable means of ensuring conservation of the plant species. However, mass propagation of medicinal plants should be accompanied with increased secondary metabolite production to guarantee their therapeutic efficacy. Therefore, the current study was aimed at understanding the different factors that affect the growth and secondary metabolite production in micropropagated E. autumnalis subspecies autumnalis. The influence of the type of gelling agent (gelrite versus agar) and source of initial/primary explant source (LDL = leaf explant derived from primary leaf regenerants and LDB = leaf explant derived from primary bulb regenerants) were evaluated. Gelrite-solidified medium significantly improved shoot proliferation when compared to the use of agar as a solidifying medium. In contrast, quantified phytochemicals such as flavonoids and phenolics were more enhanced in agar-supplemented media. On the basis of the explant source, shoot proliferation and secondary metabolites in regenerants from LDB were similar to those from LDL in most cases. Overall, the type of gelling agents and primary explant source individually or/and interactively significantly influenced the growth parameters as well as the production of iridoid, condensed tannin, flavonoid and phenolic content. The influence of different types of plant growth regulators (PGRs) on growth, phytochemical and antioxidant properties were evaluated. The PGRs were BA (benzyladenine); mT (meta-topolin); mTTHP [meta-topolin tetrahydropyran-2-yl or 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine]; MemT [meta-methoxytopolin or 6-(3-methoxybenzylamino)purine]; MemTTHP [meta-methoxy 9-tetrahydropyran-2-yl topolin or 2-[6-(3-Methoxybenzylamino)-9-(tetrahydropyran-2-yl)purine] and NAA (α-naphthalene acetic acid). Five cytokinins (CKs) at 2 μM in combination with varying (0, 2.5, 5, 10, 15 μM) concentrations of NAA were tested. After 10 weeks of in vitro growth, the regenerants were acclimatized in the greenhouse for four months. Growth, phytochemical content and antioxidant activity of in vitro regenerants and ex vitro-acclimatized plants were evaluated. The highest number of shoots (approximately 9 shoots/explant) were observed with 15 μM NAA alone or with BA treatment. Acclimatized plants derived from the 15 μM NAA treatment had the highest number of roots, largest leaf area and widest bulb diameter. While applied PGRs increased the iridoids and condensed tannins in the in vitro regenerants, total phenolics and flavonoids were higher in the PGR-free treatment. In contrast to the PGR-free regenerants, 5 μM NAA and 2 μM BA treatments produced the highest antioxidant activity in the DPPH (55%) and beta-carotene (87%) test systems, respectively. A remarkable carry-over effect of the PGRs was noticeable on the phytochemical levels and antioxidant activity of the 4-month-old plants. In addition to the development of an optimized micropropagation protocol, manipulating the type and concentration of applied PGRs may serve as an alternative approach to regulate phytochemical production in Eucomis autumnalis subspecies autumnalis. The influence of smoke-water (SW), karrikinolide (KAR1) and CK analogues (PI-55 = 6-(2-hydroxy-3-methylbenzylamino)purine and INCYDE= inhibitor of cytokinin dehydrogenase or 2-chloro-6-(3-methoxyphenyl)aminopurine) individually or in combination with some selected PGRs [BA (4 μM), NAA (5 μM) and both] for in vitro propagated E. autumnalis subspecies autumnalis was evaluated. While these compounds had no significant stimulatory effect on shoot proliferation, they influenced root length at varying concentrations and when interacted with applied PGRs. The longest roots were observed in SW (1:1500), PI-55 and INCYDE (0.01 μM) treatments. There was an increase in the concentration of quantified phytochemicals (especially condensed tannins, flavonoids and phenolics) with the use of these compounds alone or when combined with PGRs. In the presence of BA, an increase in the concentration of PI-55 significantly enhanced the condensed tannin, flavonoid and phenolic contents in the regenerants. Both phenolic and flavonoid content in E. autumnalis subspecies autumnalis were significantly enhanced with 0.01 μM INCYDE. Condensed tannins was about 8-fold higher in 10-7 M KAR1 with BA and NAA treatment when compared to the control. To some varying degree, the effect of the tested compounds on the antioxidant activity of the in vitro regenerants was also noticeable. In most cases, there was no direct relationship between the level of phytochemicals and antioxidant activity recorded. The current findings indicate the array of physiological processes influenced by SW and KAR1 during micropropagation. In addition, targeting or manipulation of phytohormone metabolic pathways using CK analogues demonstrated some noteworthy effects. Perhaps, it may offer other potential practical applications in plant biotechnology and agriculture. Thus, more studies such as quantification of endogenous hormones and identification of specific phytochemicals responsible for the bioactivity in this species will provide better insights on the mechanism of action for CK analogues as well as SW and KAR1. / M. Sc. University of KwaZulu-Natal, Durban 2014.

Dierama is a genus of plants with a potential to be developed as ornamental plants. It falls under the Iridaceae family and comprises of 44 species. Dierama erectum Hilliard, an attractive species with horticultural potential is mainly found in rough wet grasslands. Its corms are used for enemas and treating stomach ailments in southern African traditional medicine. Due to its habitat transformation by afforestation and the exploitation of its underground parts (corms) in traditional medicine, this plant is among the most vulnerable and rare species within its genus. Seed parasitism by Urodon lilli also hampers its conventional propagation. The increase in demand for ornamental and medicinal plants increases pressure on wild plant populations. Micropropagation is a useful tool for clonal propagation of plants as it does not only help in alleviating pressure on wild plants but an effective micropropagation protocol could also provide a foundation for plant genetic transformation, which could result in the development and introduction of new ornamental varieties into commercial markets. This research was aimed at developing a micropropagation protocol for D. erectum to ensure readily available source material for medicinal and horticultural use as well as serving as an alternative for its conservation. Seed decontamination and germination were successful when 0.2% HgCl2 or 2.5% NaOCl + 1% Benlate® were used. However, for safety reasons, 2.5% NaOCl + 1% Benlate® was used in all subsequent experiments. The shoot regenerative capacity of leaf, hypocotyl and root explants obtained from in vitro germinated seedlings was evaluated by culturing them individually on MS medium supplemented with various concentrations of BA. Only hypocotyl explants produced adventitious shoots. Since no shoots or callus was produced from leaf and root explants, hypocotyl explants were used in the development of a micropropagation protocol. Different types and concentrations of cytokinins (BA, mT, KIN and Z) with or without NAA were evaluated for their effect on adventitious shoot production. Maximum shoot number per explant (4.20 ±0.51) was obtained in MS medium supplemented with 1.0 ìM Z after 8 weeks. This was followed by a combination of KIN (2.0 ìM) and NAA (0.5 ìM) resulting in a production of 3.67 ± 0.81 shoots per explant. For BA treatments, the highest shoot multiplication (3.20 ± 0.22 shoots per explant) was achieved when 2.0 ìM was combined with 1.0 ìM NAA. mT gave maximum shoot production (3.09 ± 0.99 shoots per explant) when 2.0 ìM mT was combined with 2.0 ìM NAA. The effects of photoperiod and light intensity were investigated for the purpose of optimizing shoot multiplication. An average of 12.73 ± 1.03 shoots per explant were obtained after 8 weeks from shoots grown in 16 h light at a 100 ìmol m-2 s-1 light intensity. The 24 h light treatments and a light intensity lower than 100 ìmol m-2 s-1 negatively affected growth and regeneration of D. erectum. These results highlighted the need for evaluating environmental conditions when developing micropropagation protocols. Corm induction experiments were done with the intention of facilitating acclimatization of D. erectum ex vitro. Various concentrations of ancymidol, activated charcoal and sucrose did not promote in vitro corm formation, thus auxins (IAA, IBA and NAA) were tested for their efficiency in rooting. Plants rooted successfully after 8 weeks on MS medium supplemented with 1.0 ìM IBA, yielded the longest roots (4.63 ± 0.70 cm) and an average root number of 2.73 ± 0.40. All NAA treatments resulted in stunted roots. Plants grown in vitro were potted in trays containing a 1:1 ratio of soil: vermiculite and placed in the mist house for 2 weeks. They were then transferred to the greenhouse for further acclimatization. After 2 months, plants had formed corms. The largest corms (0.45 ± 0.026 cm in diameter) were found in plants pre-treated with 0.5 ìM IBA. Maximum plant survival percentage (73%) was also associated with this treatment. A successful micropropagation system for Dierama erectum was therefore developed. The utilisation of this protocol can yield about 15137 plants from one explant in a year. This will expand our existing knowledge about micropropagation of plants in the genus Dierama and will be useful in the conservation of this species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The crisis of newly emerging diseases and the resistance of many pathogens to currently used drugs, coupled with the adverse side-effects of many of these drugs have necessitated the continuous search for new drugs that are potent and efficacious with minimal or no adverse side-effects. The plant kingdom is known to contain many novel biologically active compounds, many of which could potentially have a higher medicinal value when compared to some of the current medications. Indeed, the use of plants in traditional medicine, especially in African communities, is gaining more importance due to their affordability and accessibility as well as their effectiveness. Exponential population growth rates in many developing countries has resulted in heavy exploitation of our plant resources for their medicinal values. In addition, plant habitat destruction arising from human developmental activities has contributed to the fragmentation or loss of many plant populations. Owing to these factors, many plant species with horticultural and/or medicinal potential have become either extinct or are threatened with extinction. These threatened species cut across different taxonomic categories including shrubs, trees and succulents. Without the application of effective conservation strategies, the medicinal and/or horticultural potential of such threatened species may be totally lost with time. The extinction of such species could lead to the loss of potential therapeutic compounds and/or genes capable of being exploited in the biosynthesis of new potent pharmaceutical compounds. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.